A simple new mechanistic hypothesis which could lead to an understanding of how cells sense and respond homeostatically to their cholesterol will be explored. The pool of cholesterol in the endoplasmic reticulum (ER) controls cell cholesterol accretion by modulating the activity of several downstream proteins embedded therein. The ER cholesterol pool is itself regulated by the delivery of cholesterol from the bulk pool in the plasma membrane (PM). A novel concept recently suggested by Harden McConnell is that PM cholesterol makes stoichiometric complexes with phospholipids. PM cholesterol in excess of this equivalence point is monomeric and has a high escape potential. It could therefore redistribute to the ER, setting the cholesterol level therein, and thus directing ER homeostatic activity. Human erythrocyte membranes will be used to establish this hypothesis. The pool of active cholesterol monomers will be characterized under various conditions. For example, modulation of this pool by the intercalation of other sterols, lipids and small water-soluble effectors as well as by bilayer lipid scrambling will be examined. Artificial sterol-containing lipid monolayers will be used to elucidate the molecular basis of red cell behavior. The findings with red cells will be applied to cultured human fibroblasts to test whether the ER cholesterol pool size is set by plasma membrane cholesterol monomer activity in these cells. Whether efflux of plasma membrane cholesterol to high-density lipoproteins depends on active cholesterol monomers will be evaluated. An alternative hypothesis will also be tested, namely, that plasma membrane cholesterol level is signaled to the ER by cholesterol-dependent raft formation. Whether the distribution of cholesterol monomers between the PM and intracellular membranes is governed by passive equilibration or active regulation will be tested using blockers of protein kinase C activity. Whether phosphatidylcholine biosynthesis in the ER is regulated by cholesterol monomer activity in the plasma membrane and whether the abundance of PM sphingomyelin sets the level of PM cholesterol will also be tested. Finally, high throughput multi-well plate assays for active cholesterol monomers in the PM of human fibroblasts and red cells will be used to screen for small molecules that can decrease cell cholesterol and thus serve as potential therapeutic agents against atherosclerosis.